A variety of techniques for detection of nucleic acids involve capture of the nucleic acids to a surface through hybridization of each nucleic acid to an oligonucleotide (or other nucleic acid) that is attached to the surface. For example, DNA microarray technology, which is widely used to analyze gene expression, relies on hybridization of DNA targets to preformed arrays of polynucleotides. See, e.g., Lockhart and Winzeler (2000) “Genomics, gene expression and DNA arrays” Nature 405:827-36, Gerhold et al. (2001) “Monitoring expression of genes involved in drug metabolism and toxicology using DNA microarrays” Physiol Genomics 5:161-70, Thomas et al. (2001) “Identification of toxicologically predictive gene sets using cDNA microarrays” Mol Pharmacol 60:1189-94, and Epstein and Butow (2000) “Microarray technology—enhanced versatility, persistent challenge” Curr Opin Biotechnol. 11:36-41.
A typical DNA microarray contains a large number of spots, with each spot containing a single oligonucleotide intended to hybridize to a particular nucleic acid target. For example, the GeneChip® microarray available from Affymetrix (Santa Clara, Calif.) includes thousands of spots, with each spot containing a different single 25mer oligonucleotide. Multiple (e.g., about 20) oligonucleotides that are perfect matches for a particular target nucleic acid are typically provided, with each oligonucleotide being complementary to a different region of the target nucleic acid. Additional spots including mismatch oligonucleotides having a single nucleotide substitution in the middle of the oligonucleotide are also included in the array. Since binding to a single 25mer may not result in specific capture of the target nucleic acid, statistical methods are used to compare the signals obtained from all the spots for a particular target nucleic acid (e.g., perfectly matched and mismatched oligonucleotides) to attempt to correct for cross-hybridization of other nucleic acids to those spots.
In another approach, longer probes are used to form the spots in the microarray. For example, instead of short oligonucleotides, longer oligonucleotides or cDNAs can be used to capture the target nucleic acids. Use of such longer probes can provide increased specificity, but it can also make discrimination of closely related sequences difficult.
DNA microarray technology has also been employed for enrichment of specific sequences for high throughput sequencing. Next-generation sequencing platforms facilitate large scale sequencing efforts, e.g., for detection of polymorphisms, association studies, mapping, or detection of somatic mutations. However, such efforts still require simplification of the target population, e.g., to include only a subset of the genome. While microarray hybridization can be used to prepare samples for sequencing, concerns regarding specificity are similar to those discussed for gene expression analysis. In addition, microarray-based capture can be time-consuming, require relatively large amounts of starting material, and involve additional processing steps such as enzymatic amplification of the captured DNA.
Improved methods for capturing target nucleic acids to surfaces are thus desirable. Among other aspects, the present invention provides methods that overcome the above noted limitations and permit rapid, simple, and highly specific capture of nucleic acids, including simultaneous capture of multiple nucleic acids, capture of long nucleic acids, and capture of nucleic acids for sequence analysis. A complete understanding of the invention will be obtained upon review of the following.